Protein synthesis is dynamic, with rapid reductions in translation in response to many different environmental stresses. An important mechanism for this translational control involves GCN2 protein kinase phosphorylation of eIF2 (eIF2~P), a translation initiation factor that delivers initiator Met-tRNAi(Met) to the translation machinery during the initiation phase of protein synthesis. The ensuing reduction in global translation initiation conserves energy and provides time for cells to reprogram gene expression with a focus on stress alleviation. Accompanying this global translational control, eIF2~P selectively enhances the translation of genes, such as the mammalian transcription activator ATF4 (yeast counterpart GCN4), which are important for essential adaptive functions. The idea that ATF4 is a common downstream target that integrates signaling from GCN2, and other eIF2 kinases, has led to the eIF2~P/ATF4 pathway being collectively referred to as the Integrated Stress Response (ISR). The ISR serves essential adaptive functions;however, perturbations in or unabated induction of these stress responses can contribute to morbidity. Our studies are focused on understanding the molecular mechanisms regulating protein synthesis and stress response pathways and how these processes are beneficial or adverse in a given biomedical context. This research is important for understanding the progression of many diseases, including diabetes, cancer, and cardiovascular and neurodegenerative disorders, with a long term goal of development of biomarkers and therapeutic treatments. Our hypothesis is that GCN2 recognizes different stresses and facilitates gene expression that is important for ameliorating cellular damage and treating and preventing disease. In this proposal, we will address how GCN2 recognizes and gauges environmental stresses to elicit eIF2~P and translational control, and the underlying mechanism by which can recalibrate the protein synthetic machinery, such that mRNAs are individually evaluated, leading to prescribed changes in translation efficiencies. We propose three aims. 1) Characterize the mechanisms activating GCN2 in the ISR. 2) Characterize the mechanisms and functional significance of translational control in the ISR. 3) Characterize the role of the combined translational and transcriptional regulation of ATF4 in the ISR. Completion of the proposed experiments will provide significant new insight into the molecular processes activating GCN2 and the ISR, and the complex mechanisms by which eIF2~P controls global and gene- specific translation. These fundamental findings will be important for understanding how the ISR contributes to the progression of stress-related diseases, with the promise of developing new strategies for diagnosis and treatment. PUBLIC HEALTH RELEVANCE: This proposal is focused on understanding the molecular mechanisms regulating protein synthesis and stress response pathways and how these processes are beneficial or adverse in a given biomedical context. This research is important for understanding the progression of many diseases, including diabetes, cancer, and cardiovascular and neurodegenerative disorders, with a long term goal of development of biomarkers and therapeutic treatments.